Histones are proteins which package eucaryotic DNA into chromatin. Unique among the core histone protein families, the histone H2A family comprises three subfamilies which differ by significant, evolutionarily conserved protein sequence differences. The H2A1 subfamily, which is cell cycle regulated, comprises about 80% of the bulk H2A of mammalian chromatin while the H2AX and H2AZ subfamilies, which are cell cycle independent, comprise about 10% each. During this year we have obtained some insightful results on the possible roles of histone H2AX in chromatin. The major finding during this year is that of an immediate and massive response of cells subjected to ionizing radiation through the formation of gamma components. We have identified these gamma components as histone H2AX derivatives that have been phosphorylated on serine 139. In normal human IMR90 fibroblasts and CHO cells, the amount of gamma-H2AX increases to a half maximal value in about 3 minutes, a maximal value in 10-30 minutes, followed by a slower return to near pre-irradiation values with a half-time of about 2 hr. The fraction of gamma-H2AX increases with the amount of radiation and at doses below 50 Grays appears to be linearly proportional to the amount of radiation. In IMR90 fibroblasts, about 0.6% of the H2AX is converted to gamma-H2AX forms for each Gy of ionizing radiation at doses below 50 Gy, numbers which correspond to about 1000 H2AX molecules being phosphorylated on serine-139 per induced DNA double-strand break. This finding coupled with the finding that the fraction of H2AX phosphorylated on serine-139 is independent of the relative amount of H2AX suggests a domain rather than a stoichiometric relationship between gamma-H2AX formation and radiation dose. gamma-H2AX is found in much lower amounts in the M059J human tumor line which is deficient in DNA-activated protein kinase indicating that this enzyme be the one primarily responsible for gamma-H2AX formation. These results indicate that monitoring DNA integrity is inherent in the nucleosomal level of chromatin organization and suggest that a chromosomal domain may be the basic unit of this surveillance system. This work is currently submitted. Also using recombinant human H2A histones to prepare sufficient quantities of H2AX and H2AZ to use in various structural and functional assays, we have obtained evidence that the H2AZ-H2B dimer is unstable at physiological salt conditions compared to the H2AX-H2B and H2A1-H2B dimers. We are also progressing on preparing homologous cell knockouts of H2AX and H2AZ.